The GDNF/RET/GFRa-1 comlex is involved in many physiological processes including neural migration and neuronal survival. GDNF has also been shown to ameliorate the affects of Parkinson's disease in mice, primates and, most recently, humans. Although RET signaling has been studied in some detail, the mechanism by which this signal pathway is down-regulated after ligand binding is less well understood. Down regulation is critical since it is known that RET, when activated constitutively, leads to unregulated cell growth. It will also be critical to understand, with respect to Parkinson's disease patients, how long term treatment with GDNF affects the signaling pathway and its component parts. The focus of this work will be to monitor RET and the co-receptor prior to and after GDNF treatment with respect to down regulation. Given the importance of these proteins in development, disease and the potential treatment of disease, it will be critical to determine not only how these proteins are activated but what happens after the proteins have been activated. For this project, two potential mechanisms of down-regulation will be measured. With the assistance of undergraduate and master's students, wild type and mutant RET and GFRa-1 proteins will be monitored for changes in transcription, internalization or degradation. Transcriptional regulation will be measured using northern blots and rt-PCR analysis. With respect to internalization, RE'I" and its co-receptor will be monitored using receptor labeling, internalization measurements and co-localization using confocal microscopy of both wild type and mutant proteins. Similar processes will be used to monitor degradation. Finally, once the down-regulation mechanisms have been established, the potential role of disrupted down-regulation in disease progression will be analyzed.